Mass spectrometric imaging is a technique for investigating the distribution of a substance having a specific mass by performing a mass spectrometry on each of a plurality of measurement points (micro areas) within a two-dimensional area on a sample, such as a piece of biological tissue. This technique is being developed for various applications, such as drug discoveries, biomarker discoveries, and investigation on the causes of various diseases. Mass spectrometers designed for mass spectrometric imaging are generally referred to as “imaging mass spectrometers.” This type of device may also be called by different names, such as a “microscopic mass spectrometer” or “mass microscope,” since its operation normally includes the steps of performing a microscopic observation of an arbitrary two-dimensional area on a sample, selecting a target region of the analysis based on the observed microscopic image, and performing an imaging mass spectrometry of that region. In the present specification, the term “imaging mass spectrometer” will be used. Non Patent Literatures 1 and 2 disclose the configurations of commonly known imaging mass spectrometers and examples of analyses performed with those devices.
In an imaging mass spectrometer, a set of mass spectrum data over a predetermined mass-to-charge-ratio range is obtained at each of a large number of measurement points within a two-dimensional area on a sample. In order to realize a high level of mass-resolving power, a time-of-flight mass spectrometer (TOFMS) is normally used as the mass analyzer. The amount of mass spectrum data (or time-of-flight spectrum data) per one measurement point is considerably greater than that of the mass spectrum data obtained with other types of mass spectrometers, such as a quadrupole mass spectrometer. Furthermore, to obtain a finer imaging graphic (i.e. a higher level of spatial resolution), the spacing of the measurement points needs to be reduced, which leads to an increase in the number of measurement points on a single sample. Therefore, if the mass spectrometric imaging needs to be performed with a high level of mass-resolving power and a high level of spatial resolution, the total amount of data per one sample will be enormous.
In order to create and display an imaging graphic or perform a statistical analysis by processing data with a commonly used personal computer, it is necessary to load the entire set of data to be processed into the main memory (normally, RAM) of the computer. However, the storage capacity actually available in the main memory of commonly used personal computers is limited, and it is difficult to entirely load the previously mentioned high-precision imaging mass spectrometric data. In such a case, it has been necessary to limit the range of the imaging graphic to be created and displayed in accordance with the limitation on the amount of data that can be loaded into the main memory, or to perform a process using a portion of an external storage device (e.g. hard disk drive) as a virtual main memory while accepting the decrease in the processing rate.
In view of such a problem, a technique in which mass spectrum data obtained with an imaging mass spectrometer are stored in a compressed form is disclosed in Patent Literatures 1-3. By using such a data compression technique, the imaging mass spectrometric data to be processed can be converted into a smaller size of data and loaded into the main memory. Furthermore, according to the technique disclosed in Patent Literature 1, an index showing the correspondence between the position in the array of the original mass spectrum data before compression and the position in the array of the compressed data is created and stored together with or separately from the compressed data. When a set of data (ion-intensity values) corresponding to a given mass-to-charge ratio needs to be retrieved, the compressed data corresponding to the requested data are located with reference to the index information, and the located data are decoded. By this technique, the requested data can be quickly retrieved despite the use of the compressed data.
A MALDI ion source, which is normally used in imaging mass spectrometers, is an ionization technique suitable for biological samples. However, it has the drawback that the ion intensity varies by a considerable amount for each measurement (i.e. for each shot of a laser beam). To compensate for such a drawback, in the process of acquiring a mass spectrum at one measurement point, the measurement is performed a number of times for the same measurement point and the obtained ion-intensity signals are accumulated. However, in some cases, such an accumulation cannot sufficiently cancel the influence of the variation in the ion intensity among the measurement points. Therefore, an imaging graphic which has been simply created from the ion-intensity values obtained at the respective measurement points for a specific mass-to-charge ratio does not always correctly reflect the distribution of the substance concerned. Accordingly, a technique has conventionally been proposed in which the ion-intensity values obtained at each measurement point are not simply used in the process of creating the imaging graphic, but are converted into normalized ion-intensity values according to a predetermined criterion before being used.
For example, Non Patent Literature 1 demonstrates that it is beneficial to perform a TIC or XIC normalization on imaging mass spectrometric data before creating and displaying an imaging graphic or performing a statistical analysis. TIC is the abbreviation which stands for “total ion current,” which means the sum of the ion-intensity values obtained within the entire range of mass-to-charge ratios in the mass spectra obtained at each measurement point. By the TIC normalization, the intensity value at each mass-to-charge ratio is normalized so that every measurement point will have the same TIC value. On the other hand, XIC is the abbreviation for the “extract ion current,” which means the ion-intensity value at a specified mass-to-charge ratio or the sum of the ion-intensity values within a specified range of mass-to-charge ratios in the mass spectra obtained at each measurement point. By the XIC normalization, the intensity value at each mass-to-charge ratio is normalized so that every measurement point will have the same XIC value, and therefore, it is possible to equalize the peak height at a specific mass-to-charge ratio among the measurement points.
Operators (users) often refer to an average of the mass spectra obtained at all the measurement points or at a group of measurement points within a region of interest specified by the operator, in order to determine the mass-to-charge ratio or mass-to-charge-ratio range for which an imaging graphic needs to be displayed. Such an average mass spectrum can also be beneficially created based on the ion-intensity values obtained through the TIC or XIC normalization.
However, using such normalized ion-intensity values for the creation of an imaging graphic, for the creation of an average spectrum, for a statistical analysis or for other purposes has negative attributes as follows:
For example, in the case of displaying an imaging graphic based on normalized ion-intensity values, the appearance of the imaging graphic considerably changes depending on the normalization condition, such as what type of normalization is performed (e.g. TIC or XIC normalization) and what mass-to-charge ratio (or what range of mass-to-charge ratios) is used as the reference in the case of the XIC normalization. Therefore, the operator needs to specify the normalization condition. In most cases, the actually performed task is such that the imaging graphic and/or average mass spectrum is repeatedly displayed under various normalization conditions and the operator is requested to compare them each time and determine the most appropriate normalization condition. The normalization process requires multiplying the intensity values of the mass spectrum at each measurement point by a normalization coefficient which changes with the normalization condition. Therefore, every time the normalization condition is changed, it is necessary to perform related processes, such as calculating the normalization coefficient and performing the normalization of the imaging mass spectrometric data using the new coefficient, whereby a considerable amount of time is required for the data processing. Furthermore, storing the entire set of mass spectrum data normalized under various normalization conditions is impractical since the data size will be considerably large even if the aforementioned data compression technique for the storage of mass spectrum data obtained at each measurement point is applied.
A statistical analysis for comparing imaging mass spectrometric data of a plurality of samples is performed as follows: The mass-to-charge ratio (or mass-to-charge-ratio range) of a representative peak is selected from the average mass spectrum or a maximum intensity mass spectrum which is created by extracting the maximum intensity value at each mass-to-charge ratio over the entire group of measurement points. The ion-intensity value at the selected mass-to-charge ratio in the mass spectrum at each measurement point is determined A peak matrix which shows a set of mass-to-charge-ratio values and ion-intensity values is created, and various statistical analyses are performed on this peak matrix. To correctly perform a statistical analysis, the statistical analysis may be repeated while changing the mass-to-charge-ratio value in the peak matrix. However, this significantly lowers the working efficiency, since the calculation of the peak matrix takes a considerable amount of time. The process will be even more complex and time-consuming if it includes a statistical analysis based on imaging mass spectrometric data normalized under various conditions.
Furthermore, in general, the software for creating and displaying an imaging graphic for a specific mass-to-charge ratio or an average spectrum over a specific region of interest based on imaging mass spectrometric data, and the software for performing statistical analyses have been provided as independent programs due to the limitation on the capacity of the main memory available on a computer. Therefore, for example, in order to visually check a detailed imaging graphic for a mass-to-charge ratio which has been determined to be useful by a statistical analysis, the operator needs to perform the extremely cumbersome task of interchanging a data file between the separate software programs as well as starting and ending each of those programs.